The present invention relates to a seed, a plant, a variety, an inbred, and a hybrid which contain a level of resistance to more than one herbicide. Such plants have advantages over plants having a single gene for herbicide resistance, especially with respect to prevention of resistance development in the target weed plants.
The features of a commercially competitive plant varieties generally include more than high yield with excellent standability. While yield is the single most critical input which affects the crop producer""s profit, producers expect consistency of yield from year to year, disease resistance, other value-added traits, andxe2x80x94more recentlyxe2x80x94herbicide resistance. The addition of herbicide resistance has created both opportunities as well as tremendous challenges in production agriculture.
Historically, herbicide treatments have been an integral part of modern agriculture because they provide cost-effective increases in agricultural productivity. Increased yields result from reduced weed competition for water, light, and nutrients. In addition, crop quality often improves in the absence of contaminating weed seeds. Herbicides can also aid soil conservation efforts through no-till agricultural practices, wherein herbicides rather than tillage are used to reduce weed populations prior to planting.
Herbicides generally give more consistent weed control compared to tillage in many environments. Consequently, there is increasing use of both non-selective herbicides for weed control prior to crop establishment and selective herbicides for crop weed control while a crop is growing. Non-selective herbicides kill or inhibit the growth of all actively growing plant material. Selective herbicides are those herbicides generally used for the suppression of growth of certain plant species (usually weeds), while leaving another species (usually a crop) unaffected. In North America and many other countries, these herbicides have benefitted the farmer by enabling the earlier planting of short-season crops, and by improving weed control in many cropping systems.
Herbicide development programs have always assessed the crop safety of a given herbicide in a weed control system, so that no crop yield reduction results from herbicide application. In fact, crop safety has been a focus of chemical herbicide discovery and plant breeding for the last 30 years, and thousands of chemical analogues have been screened to allow identification of herbicides that control target weeds and are safe to crops minimizing yield loss due to chemical stress. Nevertheless, while several classes of herbicides possess a broad spectrum of efficacy, many of the herbicides lack selectivity and severely injure or kill crop plants at the application rates required for effective weed control.
Examples of widely used herbicides are chlorimuron and thifensulfuron, which belong to the sulfonylurea class. They inhibit the plant enzyme acetolactate synthase (also called ALS), and soybeans which are resistant to these herbicides are referred to as STS (also called sulfonylurea tolerant) soybeans. These herbicides are the active ingredients in Classic and Pinnacle, respectively, and are registered for control of broadleaf weeds in soybeans as described in Weed Science Society of America, Herbicide Handbook, 7th edition (1994). While chlorimuron and thifensulfuron are registered for use in non-STS soybeans, they can cause significant crop injury, especially if applied post-emergence as described in Fielding and Stoller, Weed Technol. 4:264-271 (1990); Fielding and Stoller, Weed Sci. 38:172-178 (1990); Newsom and Shaw, Weed Sci. 42:608-613 (1994); and Ahrens, Weed Technol. 4:524-528 (1990). Factors which influence the extent of herbicide injury are physiological stresses from poor seed quality, delayed emergence in cold and wet soils, seedling diseases, etc.; soil pH and climatic conditions (i.e. temperature and humidity) when applications are made; and injury from prior applications of chemicals (e.g. insecticides and other herbicides).
When these herbicides are combined, either intentionally or unintentionally, positive and negative interactions can result. Chlorimuron and thifensulfuron are sold pre-mixed at elevated rates under the trade names Synchrony and Reliance, and act cooperatively to broaden the spectrum of weeds controlled. On the other hand, thifensulfuron interacts synergistically with imazethapyr (the active ingredient in the herbicide Pursuit) at normal use rates to severely injure non-STS soybeans; this combination causes less injury to STS soybeans but injury can exceed 20% which is commercially unacceptable and is discussed in Simpson and Stoller, Weed Technol. 9:582-586 (1995).
Glyphosate, which belongs to a different class of herbicide and is the active ingredient in both Roundup (also called RR) and Roundup Ultra, complements activity of the other herbicides (e.g. 2,4-D and dicamba). In some cases, glyphosate interacts synergistically with these other herbicides when they are applied in combination, as shown in Moshier, Weed Sci. 28:722-724 (1980) and Flint and Barrett, Weed Sci. 37:12-18 (1989). Tank mixing Classic at 0.5 oz/A or Pinnacle at 0.125 oz/A with Roundup at 16 fl oz/A increases control of broadleaf weeds but, in the case of Pinnacle, injury of Roundup Ready soybean is greater with the combination than with Roundup alone as discussed in Lich and Renner, Proc. NCWSS 50:124 (1995). Combination of Roundup Ultra with Synchrony (premix of chlorimuron plus thifensulfuron at elevated rates) effectively controls a broad spectrum of weeds.
Combining glyphosate with Synchrony or Reliance has the potential of increasing the spectrum of weeds (e.g. annual and perennial grasses, smartweeds, nightshade, pigweed spp., morningglory spp., etc.) that are controlled. Consequently, combining or xe2x80x9cstackingxe2x80x9d a level of resistance to both glyphosate and ALS in soybeans will allow these herbicide combinations to be used for effective weed control without crop injury.
With the development of chemical crop protection and the increasing availability of effective selective herbicides, monocultures of crops have become common. This has led to repeated application of the same or similar herbicides to these crops. More recently, in conservation or zero-tillage crop establishment systems, cultivation for weed control has largely been replaced by the use of selective and non-selective herbicides. Thus, two prevailing conditions are present in these cropping systems: (i) the frequent use of a limited range of effective herbicides and (ii) reliance upon these herbicides to the exclusion of other forms of weed control. Where these conditions prevail, herbicide-resistant weeds will increase in frequency (i.e. evolve) if there is heritable variability in response to herbicide application in weed populations and selective mortality from the herbicides.
Given the existence of genetic variation, the rate of evolution will be determined by the mode of inheritance of resistance traits, together with the intensity of selection. The evolution of resistance under persistent applications of herbicide may be considered as an example of recurrent selection in which there is a progressive, and sometimes rapid, shift in average fitness of populations of weeds exposed to herbicide. Once established, gene flow via seed distribution has probably contributed to the spread of resistant weeds. A major determinant in the selection of herbicide-resistant biotypes is the effective selection intensity that differentiates resistant individuals (more fit) from susceptible one (less fit) in the face of selection (the application of herbicide).
There are two ways in which resistance traits may arise within a weed population. A major gene, or genes may be present at low frequency, or mutate, so that selection acts to change a population which is initially susceptible. Alternatively, recurrent selection may act on continuous (quantitative) variation and achieve a progressive increase in average resistance from generation to generation, with changes in gene frequency at many loci conferring resistance.
Genetic variation can arise de novo by mutation (or recombination) or be preexisting. We can thus distinguish two situations with regard of genetic variation for herbicide resistance in nonselected populations: (1) factors affecting the acquisition of resistance by novel mutation and (2) factors affecting the probability of preexisting variation for resistance.
One of the most significant occurrences in herbicide resistance has been the advent of weeds resistant to herbicides that inhibit acetolactate synthase (ALS). This is because ALS inhibitor herbicides have become extremely important new tools in agricultural production, and any development which might limit their utility is regarded as serious. The use of two major classes of ALS inhibitor herbicides alonexe2x80x94sulfonylureas and imidazolinonesxe2x80x94has grown to a 1991 market value of approximately $1.3 billion. This popularity is due to relatively low use rates, sound environmental properties, low mammalian toxicity, wide crop selectivity, and high efficacy. Five years after the initial use of an ALS inhibitor herbicide, the first resistant weeds appeared, and their incidence has steadily increased both in number of sites and species. A large factor in the appearance of resistance is the high selection pressure imposed by ALS inhibitor herbicides on very sensitive weed species. The occurrence of target site, ALS inhibitor resistance as most frequently resulted form the selection pressure associated with long residual herbicides and monoculture or near monoculture conditions.
When weed populations become sufficiently enriched with weed resistant biotypes such that they cannot be controlled by the usual rate of herbicide and the weed burden causes, or threatens, loss of crop production, then changes in weed control techniques must be implemented. However, in order to minimize the need for remedial measure after resistance develops, more complex strategies for weed control are required in order to delay or prevent the evolution of resistance. Ideally, these strategies should include the use of crop rotations, herbicide mixtures or rotations, tillage, and integrated pest management techniques where possible.
For a number of technical and practical reasons, resistance to herbicides in agronomically important crops was among the first traits to which recombinant DNA technology and novel genetic approaches were applied. The advent of Roundup Ready (RR) Soybeans which have a level of resistance to glyphosate, and Liberty Link (LL) Soybeans which have a level of resistance to the herbicide glufosinate has provided new and exciting opportunities in agriculture. This technology has allowed developers of soybean varieties to build herbicide selectivity and true crop safety mechanisms into soybean. This approach thus has expanded the utility of proven, previously non-selective, broad spectrum herbicides. These herbicide resistant crops enable improved weed control and greater flexibility in herbicide application, resulting in better production systems. New herbicide resistance traits can be developed as components of new weed control systems featuring herbicides with the beneficial environmental characteristics needed to meet current and future rigorous demands on active ingredients.
Unfortunately economic and/or governmental regulations often limit the implementation of these strategies. For example the predominant use of one herbicide in monoculture as practiced in the northern Great Plains is among the least appropriate from a weed resistance management perspective. Many agronomic practices are between the extremes of monoculture and complex rotations, and include both monoculture with herbicides having several modes of action, and rotational culture with herbicides having single modes of action. Even these more integrated markets, however, are tending towards less herbicide diversity and an increased weed resistance potential due to the introduction of new herbicides with the same chemistry and new soybean herbicide resistant crops (HRC).
The present invention relates to a seed, a plant, a variety, an inbred, a hybrid and the progeny derived from them.
More specifically, the invention relates to a plant having resistance to at least two herbicides. The invention further relates to a plant having resistance to at least two herbicides and having a commercially acceptable grain yield. The invention further relates to a method of producing a hybrid plant.
In another aspect, the invention relates to a method of producing a hybrid soybean seed. The present invention further relates to a soybean plant having resistance to at least two herbicides. The invention further relates to a soybean plant having resistance to at least two herbicides and having a commercially acceptable grain yield.
The invention further relates to a soybean plant having a level of resistance to glyphosate or Roundup(trademark) herbicide and to sulfonylurea or STS(trademark) herbicide.
The invention further relates to a soybean plant having a level of resistance to glufosinate or Liberty(trademark) herbicide and sulfonylurea or STS(trademark) herbicides.
The invention further relates to a soybean plant having a level of resistance to glyphosate and to glufosinate herbicides.
The invention further relates to a soybean plant having a level of resistance to glyphosate or Roundup(trademark), glufosinate or Liberty(trademark) and sulfonylurea or STS(trademark) herbicide.
The present invention further relates to a soybean plant having a level of resistance to at least two herbicides, wherein said herbicides are selected from the group consisting of atrazine, ALS inhibitor, glyphosate, glufosinate and isoxoflutole.
The present invention further relates to a method of producing a soybean plant having resistance to at least two herbicides.
To provide an understanding of several of the terms used in the specification and claims, the following definitions are provided:
Agronomically acceptable iniuryxe2x80x94As used herein the term agronomically acceptable injury means any herbicide injury resulting in a less than 10 percent reduction in yield when compared to the same variety having no herbicidal injury.
Agronomically Fitxe2x80x94As used herein, the term agronomically fit means a genotype that has the culmination of many distinguishable traits such as emergence, vigor, vegetative vigor, disease resistance, seed set, standability and threshability which allows a producer to harvest a product of commercial significance.
ALS Inhibitorxe2x80x94As used herein, the ALS inhibitor means any herbicidally effective form of sulfonylureas, triazolopyrimidine sulfonamides, imidazolinones or heteroaryl ethers including any salt thereof or other related compounds or derivatives.
Atrazinexe2x80x94As used herein, the term atrazine means any herbicidally effective form of triazine, including 6-40 N-ethyl-Nxe2x80x2-(1 methylethyl)-1,3,5 triazine-2,4 diamine, and including any salt thereof or other related compounds or derivatives.
Base Populationxe2x80x94As used herein, the term base population means the development of segregating populations during inbreeding until the desired lelvel of homozygosity is achieved. Selection of plants or parts of plants are either random or nonrandom and advanced to the next generation. The lines derived from the plants are evaluated for the characteristics of interest.
Commercially acceptablexe2x80x94The term commercially acceptable means a soybean variety having a grain yield of greater than 35 bushels per acre over at least two years and 10 environments.
Glufosinatexe2x80x94As used herein, the term glufosinate means any herbicidally effective form of phosphinothricin, including any salt thereof or other related compounds or derivatives.
Glyphosatexe2x80x94As used herein, the term glyphosate means any herbicidally effective form of N-phosphonomethylglycine including any salt thereof or other related compounds or derivatives or any other 5-enolpyrunyl 3-shilkimate phosphate synthase inhibitor.
Herbicide Resistancexe2x80x94The term herbicide resistance means the ability to survive with agronomically acceptable injury, a concentration of herbicide that is normally lethal or extremely injurious to individual plants of a given species.
Isoxaflutolexe2x80x94As used herein, the term isoxaflutole means any herbicidally effective form of 5-cyclopropyl-4 (methane sulphonyl 1-4-thifluoromethylbenzoyl), isoxazole or other related compounds or derivatives.
Stackingxe2x80x94As used herein, the term stacking means genetically combining multiple herbicide resistant traits into a commercially acceptable cultivar using conventional plant breeding and/or genetic engineering methods.
The advent of genetic engineering has provided agricultural industry with soybeans that are resistant to glyphosate or glufosinate or sulfonylureas. Prior to the instant invention, a soybean variety has never been developed having more than one herbicide resistance trait combined into one soybean genotype. These herbicide genes have not previously been stacked in any commercial or wild type soybean. Having multiple herbicide resistant genes in one soybean variety substantially expands the utility to use proven, previously nonselective, broad spectrum herbicides. Herbicide Resistant Crops, also called HRC, provide improved weed control and greater flexibility in herbicide application resulting in better production systems. The use of herbicides with alternate modes of action in a given weed management system offers advantages in extending the lives of current programs, such as in weed resistance management. Also, new weed control systems featuring herbicides with beneficial environmental characteristics are needed to meet current and future rigorous demands on active ingredients.
The likelihood of target site resistance developing in a population to a mixture of herbicides is the mathematical product of the frequency of any genes conferring resistance to the mixture components. Mixtures can, therefore, be an effective tool in weed resistance management of autogamous species, because individual plants bearing multiple mutations will be extremely rare and gene flow between surviving plants is low.
Since in the instant invention the new herbicide resistance genes are introduced into varieties with acceptable genetic backgrounds (i.e. with high yield, excellent standability, and multiple disease and pest resistance), the resistances can be rapidly combined, developed into commercially acceptable cultivars, and made available to the farmer. Thus, these cultivars with stacked resistance traits can be extremely useful in agriculture.
All crop species are grown for the purpose of harvesting some product of commercial significance. Enhancement of productivity or yield of that product is a major goal of most plant breeding programs. The highest priority in most soybean cultivar development programs is increasing seed yield. Seed yield is a quantitative character controlled by many genes and strongly influenced by the environment. The heritability of yield is the lowest and the most variable of the major agronomic traits considered in cultivar development, with heritability estimates ranging from 3 to 58%. Yield is an example of a quantitative character that breeders attempt to improve beyond the level of that present in current cultivars. Disease resistance is required in most cases to protect the yield potential of a cultivar.
It is a difficult challenge to incorporate one herbicide resistant or tolerant trait into high yielding cultivars. The difficulty of obtaining a commercially acceptable variety is increased by several orders of magnitude if a breeder attempts to combine two herbicide resistance or tolerance traits into one cultivar. For a plant breeder to find a cultivar with sufficient merit (e.g. high yielding) to be increased and commercially distributed, it is necessary to make many crosses and grow thousands of experimental genotypes. The evaluation of so many genotypes is a huge task, and consumes an enormous amount of the plant breeder""s time and budget. In some instances, it can take a decade or more from the time the original cross is made to the time when a commercially viable genotype is identified.
The effectiveness of selecting for genotypes with the traits of interest (e.g., high yield, disease resistance, herbicide resistance) in a breeding program will depend upon: 1) the extent to which the variability in the traits of interest of individual plants in a population is the result of genetic factors and is thus transmitted to the progenies of the selected genotypes; and 2) how much the variability in the traits of interest (yield, disease traits, herbicide resistance) among the plants is due to the environment in which the different genotypes are growing. The inheritance of traits ranges from control by one major gene whose expression is not influenced by the environment (i.e., qualitative characters) to control by many genes whose effects are greatly influenced by the environment (i.e., quantitative characters). Breeding for quantitative traits is further characterized by the fact that: 1) the differences resulting from the effect of each gene are small, making it difficult or impossible to identify them individually; 2) the number of genes contributing to a character is large, so that distinct segregation ratios are seldom if ever obtained; and 3) the effects of the genes may be expressed in different ways based on environmental variation. Therefore, the accurate identification of transgressive segregants or superior genotypes with the traits of interest is extremely difficult and its success is dependent on the plant breeder""s ability to minimize the environmental variation affecting the expression of the quantitative character in the population. The likelihood of identifying a transgressive segregant is greatly reduced as the number of traits combined into one genotype is increased. For example, if a cross is made between cultivars differing in three complex characters, such as yield, disease resistance and herbicide resistance, it is extremely difficult to recover simultaneously by recombination the maximum number of favorable genes for each of the three characters into one genotype. Consequently, all the breeder can generally hope for is to obtain a favorable assortment of genes for the first complex character combined with a favorable assortment of genes for the second character into one genotype in addition to a herbicide resistant gene.
The methods used in cultivar development programs and their probability of success are dependent on the number of characters to be improved simultaneously, such as, seed yield, disease resistance, and herbicide resistant/tolerant traits. The proportion of desired individuals for multiple characters in a population is obtained by multiplying together the proportion of desired individuals expected in the population for each character to be improved. This assumes that the characters are inherited independently, i.e., are not genetically linked.
These principles can be applied not only to traditionally bred lines, but to transgenic lines as well. Whether combining desirable traditional and transgenic traits via hybridization of transgenic lines or co-transformation of multiple genes into one line, the combined effect on yield are likely to be multiplicative. For example, if the probability that suitable yields and disease resistance are found in 1% of lines transformed with a single herbicide resistance gene, then the probability that combining three herbicide resistance genes in a line with suitable yield and disease resistance ought to be 0.01xc3x970.01xc3x970.01 or 1xc3x9710xe2x88x926.
The likelihood of identifying a line with a suitable combination of traits is further reduced when considering the potential effects of a transgene on the regulation of metabolism within a plant. For example, one can consider the potential effect of genes conferring resistance to glyphosate, glufosinate, and sulfonylureas or imidazolanones. The genes conferring these traits are, respectively, a gene encoding the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a transgene encoding the enzyme phosphinothricin acetyl transferase (PAT), and a gene encoding a mutant acetolactate syntase (ALS) enzyme. The biochemical steps affected by these genes is illustrated in Scheme 1. In the case of PAT, the effect is not to catalyze the reaction shown, but to counteract the effect of phosphinothricin, which inhibits the enzyme glutamine synthase. Two of the genes (EPSPS and ALS) affect closely related biochemical reactions in the synthesis of amino acids.
In commercial versions of plants which are resistant to glyphosate, a gene is introduced which is controlled by the cauliflower mosaic virus 35S RNA promoter. Because this promoter is constitutively expressed, the transgenic enzyme is not under the same controls as the endogenous EPSPS promoter. Consequently, the flow of carbon through the reaction catalyzed by EPSPS would not be subject to the regulatory mechanisms which would normally be present. Thus, the amount of PEP (which can be assumed, along with other substrates normally in the cell, to be at sub-saturating levels) going through the pathway to phenylalanine, tyrosine, and tryptophan would probably be greater than normal, and could result in a shortage of PEP necessary for the production of valine, 
leucine, and isoleucine through the pathway involving ALS. This could be the reason for our observations that many lines transformed with EPSPS are not acceptable with respect to yield, and many must be screened for an acceptable line to be selected. Presumably these few acceptable lines have background genotypes which compensate for or are unaffected by the perturbations caused by the introduced gene.
Similar scenarios can be envisioned for the selection of ALS mutants resistant to sulfonylureas and transgenic lines tolerant to Liberty(trademark) herbicide. In the case of lines containing the ALS mutants, characteristics of the mutant enzyme (either Km and/or Vmax) may have been affected. In the case of lines containing the introduced PAT gene, energy resources must be diverted to produce the new enzyme at levels suitable to confer herbicide tolerance.
When these lines are combined by breeding, the background genotypes which have adjusted to the introduced or mutant genes are combined, and new genotypes must be selected. Likewise, when the genes are introduced by transformation together, the adjustments made by the background genotype must occur as well. Therefore, the frequency of genotypes with suitable yield will be reduced accordingly. Surprisingly, we have been able to select genotypes with unaffected yield resulting from the crossing of lines transformed with single genes.
Using similar techniques and other techniques well known in the art, resistance genes to other herbicides can also be combined. The instant invention relates to herbicide resistance genes, constructs, promoters and methods of incorporating the resistance genes into commercial inbreds, hybrids and varieties of many plant crops including but not limited to, the crops of corn, cotton, soybeans, canola, sunflowers, sorghum, wheat, barley, triticales, alfalfa, tomato, pepper, broccoli, rose, impatiens, carnation, geranium and petunia. Suitable genes, promoters and methods may be found in Herbicide-Resistant Crops, Editor Stephen O. Duke, CRC Lewis Publishers, 1996; Herbicide Resistance in Plants, Editors Stephen B. Powles and Joseph A. M. Holtum, CRC Press Inc., 1994; and in U.S. Pat. No. 5,084,082; 5,359,142; 5,322,938; 5,424,200; 5,164,316; 5,352,605; 5,094,945; 4,535,600 and 4,940,835, all of which are incorporated herein by reference. While it is very difficult to develop the first commercially acceptable inbred or variety having resistance to more than one herbicide, once an elite inbred or variety is produced then the combined herbicide resistance characteristic can be readily transferred to other inbreds, hybrids and varieties with appropriate backcross and selection to maintain the desirable traits.
As used herein, the term xe2x80x9cplantxe2x80x9d includes plant cells, plant protoplasts, plant cells of tissue culture from which soybean plants can be regenerated, plant calli, plant clumps, and plant cells that are intact plants or parts of plants such as pollen, flowers, seeds, leaves, stems and the like.